Rapid effects of estradiol on male aggression depend on photoperiod in reproductively non-responsive mice

In three genuses and four species of rodents, housing in winter-like short days (8L:16D) increases male aggressive behavior. In all of these species, males undergo short-day induced regression of the reproductive system. Some studies, however, suggest that the effect of photoperiod on aggression may be independent of reproductive responses. We examined the effects of photoperiod on aggressive behavior in California mice (Peromyscus californicus), which do not display reproductive responsiveness to short days. As expected, short days had no effect on plasma testosterone. Estrogen receptor alpha and estrogen receptor beta immunostaining did not differ in the lateral septum, medial preoptic area, bed nucleus of the stria terminalis, or medial amygdala. However, males housed in short days were significantly more aggressive than males housed in long days. Similar to previous work in beach mice (Peromyscus polionotus), estradiol rapidly increased aggression when male California mice were housed in short days but not when housed in long days. These data suggest that the effects of photoperiod on aggression and estrogen signaling are independent of reproductive responses. The rapid action of estradiol on aggression in short-day mice also suggests that nongenomic mechanisms mediate the effects of estrogens in short days.     

Steroid hormone mediation of limbic brain plasticity and aggression in free-living tree lizards, Urosaurus ornatus

The neural mechanisms by which steroid hormones regulate aggression are unclear. Although testosterone and its metabolites are involved in both the regulation of aggression and the maintenance of neural morphology, it is unknown whether these changes are functionally related. We addressed the hypothesis that parallel changes in steroid levels and brain volumes are involved in the regulation of adult aggression. We examined the relationships between seasonal hormone changes, aggressive behavior, and the volumes of limbic brain regions in free-living male and female tree lizards (Urosaurus ornatus). The brain nuclei that we examined included the lateral septum (LS), preoptic area (POA), amygdala (AMY), and ventromedial hypothalamus (VMH). We showed that the volumes of the POA and AMY in males and the POA in females vary with season. However, reproductive state (and thus hormonal state) was incompletely predictive of these seasonal changes in males and completely unrelated to changes in females. We also detected male-biased dimorphisms in volume of the POA, AMY, and a dorsolateral subnucleus of the VMH but did not detect a dimorphism between alternate male morphological phenotypes. Finally, we showed that circulating testosterone levels were higher in males exhibiting higher frequency and intensity of aggressive display to a conspecific, though brain nucleus volumes were unrelated to behavior. Our findings fail to support our hypothesis and suggest instead that plasma testosterone level covaries with aggression level and in a limited capacity with brain nucleus volumes but that these are largely unrelated relationships.     

Combined effects of DHEA and fadrozole on aggression and neural VIP immunoreactivity in the non-breeding male song sparrow

The male Song Sparrow, Melospiza melodia morphna, shows high levels of aggression in its non-breeding season, concomitant with basal levels of circulating testosterone (T) and estradiol (E(2)). However, administration of fadrozole, an aromatase inhibitor, decreases non-breeding aggression in the field. Circulating levels of dehydroepiandrosterone (DHEA), an androgen/estrogen precursor, correspond to the seasonal expression of aggression in this species, with high levels in the breeding and non-breeding seasons when aggression is also high, and lower levels during the molt when aggression is low. We test the hypothesis that circulating DHEA up-regulates non-breeding aggression via an aromatase-mediated mechanism. We also hypothesize that this up-regulation of aggression is partially mediated by changes in vasoactive intestinal polypeptide (VIP) in the lateral extent of the bed nucleus of the stria terminalis (BSTl) and lateral septum (LS). Birds were administered either DHEA, fadrozole, or both for 2 weeks and tested for aggression in a lab-based paradigm. As predicted, birds given DHEA were significantly more aggressive. However, fadrozole did not block this effect, and, when administered without DHEA, also led to increased aggression over controls. These results may be explained by the fact that the behaviors measured in field tests, which include more direct attack behaviors, may be under different hormonal regulation than the behaviors measured in the lab paradigm, which represent warning, or threat, behaviors. VIP immunoreactivity (VIP-ir) changed across multiple brain regions with this treatment regimen, most notably in the LSO/VFI subdivision of the lateral septum.     

Alterations in the anterior hypothalamic dopamine system in aggressive adolescent AAS-treated hamsters

Anabolic androgenic steroid (AAS) treatment throughout adolescence facilitates offensive aggression in male Syrian hamsters (Mesocricetus auratus). The present study was conducted to investigate the role of the dopaminergic system in the modulation of AAS-induced aggressive behavior. Hamsters were administered AAS during adolescence, scored for offensive aggression using the resident-intruder paradigm, and then examined for alterations in DA immunoreactivity in brain regions implicated in the aggressive phenotype, including the anterior hypothalamus (AH), the bed nucleus of the stria terminalis (BNST), the medial and central amygdala (MeA and CeA), the lateral septum (LS) and the ventrolateral hypothalamus (VLH). When compared with non-aggressive sesame-oil-treated controls, aggressive AAS-treated animals showed increased tyrosine hydroxylase immunoreactivity in anterior hypothalamic subnuclei, namely the nucleus circularis (NC) and medial supraoptic nucleus (mSON). In addition, AAS-treated animals showed altered D₂ receptor expression in the AH and the VLH, as measured by D₂-immunoreactivity. Together these results suggest that alterations in DA synthesis and function together with modifications in D₂ receptor expression in the AH may underlie neuroplastic events which facilitate AAS-induced aggression.     

Neuroendocrine correlates of behavioral polymorphism in white-throated sparrows

Interspecific differences in the neuropeptide systems of the lateral septum (LS) often parallel differences in social behavior. In rodents, some closely related species that differ in aggressive behavior also differ according to the level of vasopressin (VP) innervation of the LS. In songbirds, the neuropeptides vasotocin (VT) and vasoactive intestinal peptide (VIP) affect aggression when administered directly to the LS. Here, we tested whether the density of VT or VIP innervation of the LS reflects patterns of intraspecific behavioral polymorphism in male and female white-throated sparrows (Zonotrichia albicollis), in which the "white-stripe" (WS) morph behaves more aggressively than the "tan-stripe" (TS) morph. We found that the WS birds had more VT-immunoreactivity (IR) than the TS birds in the ventrolateral subdivision of the caudal LS (LSc.vl) and in the medial portion of the bed nucleus of the stria terminalis (BSTm). In addition, the TS birds had more densely stained VIP-IR in the LSc.vl than the WS birds. Males had more VT-IR than females in the LSc.vl and BSTm, and more VIP-IR in the LSc.vl. We also report sex and morph differences in VIP-IR in the basal hypothalamus, where VIP is synthesized and released into the portal vasculature. Males had nearly twice as many VIP-immunoreactive (ir) neurons in the infundibular nucleus than did females, and birds of the WS morph had more densely stained VIP-IR in the median eminence than TS birds. Our results support the hypothesis that differences in these neuropeptide systems underlie inter- and intraspecific differences in social behavior across vertebrates.     

Genotype/age interactions on aggressive behavior in gonadally intact estrogen receptor beta knockout (betaERKO) male mice

Estrogen, as an aromatized metabolite of testosterone, has a facilitatory effect on male aggressive behavior in mice. Two subtypes of estrogen receptors, alpha (ER-alpha) and beta (ER-beta), in the brain are known to bind estrogen. Previous studies revealed that the lack of ER-alpha gene severely reduced the induction of male aggressive behavior. In contrast, mice that lacked the ER-beta gene tended to be more aggressive than wild type (WT) control mice, although the behavioral effects of ER-beta gene disruption were dependent on their social experience. These findings lead us to hypothesize that estrogen may facilitate aggression via ER-alpha whereas it may inhibit aggression via ER-beta. In the present study, we further investigated the role of ER-beta in the regulation of aggressive behavior by examining developmental changes starting at the time of first onset, around the age of puberty. Aggressive behaviors of ER-beta gene knockout (betaERKO) mice were examined in three different age groups, puberty, young-adult, and adult. Each mouse was tested every other day for three times in a resident-intruder paradigm against olfactory bulbectomized intruder mice and their trunk blood was collected for measurements of serum testosterone after the completion of the study. Overall, betaERKO mice were significantly more aggressive than WT. These genotype differences were more pronounced in puberty and young adult age groups, but not apparent in the adult age group, in which betaERKO mice were less aggressive than those in two younger age groups. Serum testosterone levels of betaERKO mice were significantly higher than those of WT mice only in the pubertal age group, but not in young adult (when betaERKO mice were still significantly more aggressive than WT mice) and adult (when no genotype differences in aggression were found) age groups. These results suggest that ER-beta mediated actions of gonadal steroids may more profoundly be involved in the inhibitory regulation of aggressive behavior in pubertal and young adult mice.     

Species and individual differences in juvenile female alloparental care are associated with oxytocin receptor density in the striatum and the lateral septum

The neuropeptide oxytocin has been implicated in the regulation of affiliative behavior and maternal responsiveness in several mammalian species. Rodent species vary considerably in the expression of juvenile alloparental behavior. For example, alloparental behavior is spontaneous in juvenile female prairie voles (approximately 20 days of age), takes 1-3 days of pup exposure to develop in juvenile rats, and is nearly absent in juvenile mice and meadow voles. Here, we tested the hypothesis that species differences in pup responsiveness in juvenile rodents are associated with oxytocin receptor (OTR) density in specific brain regions. We found that OTR density in the nucleus accumbens (NA) is highest in juvenile prairie voles, intermediate in juvenile rats, and lowest in juvenile mice and meadow voles. In the caudate putamen (CP), OTR binding was highest in prairie voles, intermediate in rats and meadow voles, and lowest in mice. In contrast, the lateral septum (LS) shows an opposite pattern, with OTR binding being high in mice and meadow voles and low in prairie voles and rats. Thus, alloparental responsiveness in juvenile rodents is positively correlated with OTR density in the NA and CP and negatively correlated with OTR density in the LS. We then investigated whether a similar receptor-behavior relationship exists among juvenile female prairie voles by correlating individual variation in alloparental behavior with variation in OTR density. The time spent adopting crouching postures, the most distinctive component of alloparental behavior in juveniles, was positively correlated with OTR density in the NA (r = 0.47) and CP (r = 0.45) and negatively correlated with OTR density in the lateral septum (r = -0.53). Thus, variation in OTR density in the NA, CP, and LS may underlie both species and individual differences in alloparental care in rodents.     

Induction of male-typical aggression by androgens but not by estrogens in adult female mice

Ovariectomized adult CF-1 female mice were implanted with silastic capsules containing either testosterone (T), dihydrotestosterone (DHT), methyltrienolone (R1881), estradiol (E2), diethylstilbestrol (DES), or oil vehicle and were tested for aggressive behavior. The androgenic treatments (T, DHT, R1881) were highly effective in promoting male-like aggression while the estrogens (DES, E2) were completely ineffective. Subsequent receptor-binding studies confirmed assumptions about the specificity of DES, DHT, and R1881 binding to estrogen and androgen receptors in mouse hypothalamus.     

Estrogen involvement in social behavior in rodents: Rapid and long-term actions

This article is part of a Special Issue (“Estradiol and cognition”).Estrogens have repeatedly been shown to influence a wide array of social behaviors, which in rodents are predominantly olfactory-mediated. Estrogens are involved in social behavior at multiple levels of processing, from the detection and integration of socially relevant olfactory information to more complex social behaviors, including social preferences, aggression and dominance, and learning and memory for social stimuli (e.g. social recognition and social learning). Three estrogen receptors (ERs), ERα, ERβ, and the G protein-coupled ER 1 (GPER1), differently affect these behaviors. Social recognition, territorial aggression, and sexual preferences and mate choice, all requiring the integration of socially related olfactory information, seem to primarily involve ERα, with ERβ playing a lesser, modulatory role. In contrast, social learning consistently responds differently to estrogen manipulations than other social behaviors. This suggests differential ER involvement in brain regions important for specific social behaviors, such as the ventromedial and medial preoptic nuclei of the hypothalamus in social preferences and aggression, the medial amygdala and hippocampus in social recognition, and the prefrontal cortex and hippocampus in social learning. While the long-term effects of ERα and ERβ on social behavior have been extensively investigated, our knowledge of the rapid, non-genomic, effects of estrogens is more limited and suggests that they may mediate some social behaviors (e.g. social learning) differently from long-term effects. Further research is required to compare ER involvement in regulating social behavior in male and female animals, and to further elucidate the roles of the more recently described G protein-coupled ERs, both the GPER1 and the Gq-mER.     

Sexual differentiation of androgen-sensitive and estrogen-sensitive regulatory systems for aggressive behavior

CF-1 female mice were treated with either testosterone (T), diethylstilbestrol (DES), or methyltrienolone (R1881) on the day of birth and were subsequently tested for their responsiveness to the aggression-promoting property of androgen or estrogen during adulthood. The results showed that neonatal exposure to androgen enhanced subsequent sensitivity to androgenic stimulation but did not alter responsiveness to estrogens. Neonatal estrogen treatment established the capacity to exhibit aggression in response to estrogenic stimulation in adulthood but had little effect on responsiveness to androgens. These data indicate that the androgenic and estrogenic metabolites of T have distinct roles in masculinization of the neural substrate for aggressive behavior.     

Heightened aggressive behavior in mice with lifelong versus postweaning knockout of the oxytocin receptor

Previous work implicating the neuropeptide oxytocin (Oxt) in the neural regulation of aggression in males has been limited. However, there are reports of heightened aggression in Oxt knockout and Oxt receptor (Oxtr) knockout male mice when they are born to null mutant mothers; suggesting that intrauterine exposure to Oxt may be important to normal aggression in adulthood. To explore this, we examined aggression in two lines of Oxtr mice, a total knockout (Oxtr-/-), in which the Oxtr gene is absent from the time of conception, and a predominantly forebrain specific knockout (Oxtr FB/FB), in which the Oxtr gene is not excised until approximately 21-28days postnatally. Aggression was measured in males from both lines, as well as control littermates, using a resident-intruder behavioral test. Consistent with previous reports, male Oxtr-/- mice had elevated levels of aggression relative to controls. Oxtr FB/FB mice on the other hand displayed levels of aggression similar to control animals. In addition, following a resident-intruder test, Oxtr+/+ mice that displayed aggression had less c-fos immunoreactivity in the ventral portion of the lateral septum than those that did not. Further, Oxtr-/- mice had increased c-fos immunoreactivity in the medial amygdala relative to controls. These data suggest that Oxt may play an important role during development in the organization of the neural circuits that underlie aggressive behavior in adulthood, with its absence resulting in heightened aggression.     

Seasonal differences in the hormonal control of territorial aggression in free-living European stonechats

In birds, territorial aggression during the breeding season is regulated by testosterone (T). However, many bird species also express aggressive behavior during the nonbreeding season, when plasma levels of T are low. It has been suggested that during this period estrogens might play a major role in regulating territorial aggression. In the present study we compared the effects of simultaneous blockage of androgenic and estrogenic actions on territorial aggression during the breeding and nonbreeding seasons in free-living male European stonechats (Saxicola torquata rubicola). European stonechats are of particular interest since they establish territories and form pairs during both the breeding and the nonbreeding seasons. Thus territorial aggression and its endocrine control can be compared between reproductive and non-reproductive contexts. Inhibition of androgenic and estrogenic actions by simultaneous application of Flutamide and ATD reduced territorial aggression during the breeding season, but not during the nonbreeding season. Our results show that androgens and/or estrogens are involved in the endocrine control of territorial aggression in stonechats only in a reproductive context, but not in a non-reproductive one.     

Repeated anabolic-androgenic steroid treatment during adolescence increases vasopressin V(1A) receptor binding in Syrian hamsters: correlation with offensive aggression

Repeated anabolic-androgenic steroid treatment during adolescence increases hypothalamic vasopressin and facilitates offensive aggression in male Syrian hamsters (Mesocricetus auratus). The current study investigated whether anabolic-androgenic steroid exposure during this developmental period influenced vasopressin V(1A) receptor binding activity in the hypothalamus and several other brain areas implicated in aggressive behavior in hamsters. To test this, adolescent male hamsters were administered anabolic steroids or sesame oil throughout adolescence, tested for offensive aggression, and examined for differences in vasopressin V(1A) receptor binding using in situ autoradiography. When compared with control animals, aggressive, adolescent anabolic steroid-treated hamsters showed significant increases (20-200%) in the intensity of vasopressin V(1A) receptor labeling in several aggression areas, including the ventrolateral hypothalamus, bed nucleus of the stria terminalis, and lateral septum. However, no significant differences in vasopressin V(1A) receptor labeling were found in other brain regions implicated in aggressive responding, most notably the lateral zone from the medial preoptic area to anterior hypothalamus and the corticomedial amygdala. These data suggest that adolescent anabolic steroid exposure may facilitate offensive aggression by increasing vasopressin V(1A) receptor binding in several key areas of the hamster brain.     

Paternal behavior influences development of aggression and vasopressin expression in male California mouse offspring

Parental care has been demonstrated to have important effects on offspring behavioral development. California mice (Peromyscus californicus) are biparental, and correlational evidence suggests that pup retrieving by fathers has important effects on the development of aggressive behavior and extra-hypothalamic vasopressin systems. We tested whether retrievals affected these systems by manipulating paternal retrieval behavior between day 15 and 21 postpartum. Licking and grooming behavior affect behavioral development in rats, so we also experimentally reduced huddling and grooming behavior by castrating a subset of fathers. Experimentally increasing the frequency of paternal pup retrieving behavior decreased attack latency in resident-intruder in both male and female adult offspring, whereas experimental reduction of huddling and grooming had no effect. In a separate group of male offspring, we examined vasopressin immunoreactivity (AVP-ir) in two regions of the posterior bed nucleus of the stria terminalis (BNST): the dorsal fiber tracts (dBNST) and the ventral cell body-containing region (vBNST). Experimentally increasing retrievals led to an apparent shift in AVP-ir distribution. Specifically, offspring from the high retrieval group had more AVP-ir than offspring from the sham retrieval group in the dBNST, whereas the opposite was observed in the vBNST. Experimental reduction of paternal grooming was associated with increased AVP-ir in the paraventricular nucleus and also increased corticosterone and progesterone, similar to observed effects of maternal grooming on HPA function. This study provides further evidence that paternal behavior influences the development of aggression and associated neural substrates.     

Glutamic acid decarboxylase (GAD65) immunoreactivity in brains of aggressive, adolescent anabolic steroid-treated hamsters

Chronic anabolic-androgenic steroid (AAS) treatment during adolescence facilitates offensive aggression in male Syrian hamsters (Mesocricetus auratus). The current study assessed whether adolescent AAS exposure influenced the immunohistochemical localization of glutamic acid decarboxylase (GAD65), the rate-limiting enzyme in the synthesis of gamma-aminobutyric acid (GABA), in areas of hamster brain implicated in aggressive behavior. Hamsters were administered high dose AAS throughout adolescence, scored for offensive aggression, and then examined for differences in GAD65 puncta to regions of the hamster brain important for aggression. When compared with control animals, aggressive AAS-treated hamsters showed significant increases in the area covered by GAD65 immunoreactive puncta in several of these aggression regions, including the anterior hypothalamus, ventrolateral hypothalamus, and medial amygdala. Conversely, aggressive AAS-treated hamsters showed a significant decrease in GAD65-ir puncta in the lateral septum when compared with oil-treated controls. However, no differences in GAD65 puncta were found in other aggression areas, such as the bed nucleus of the stria terminalis and central amygdala. Together, these results support a role for altered GAD65 synthesis and function in adolescent AAS-facilitated offensive aggression.     

Aromatase activity in the rat brain: Hormonal regulation and sex differences

The intracellular conversion of testosterone to estradiol by the aromatase enzyme complex is an important step in many of the central actions of testosterone. In rats, estrogen given alone, or in combination with dihydrotestosterone, mimics most of the behavioral effects of testosterone, whereas treatment with antiestrogens or aromatase inhibitors block facilitation of copulatory behavior by testosterone. We used a highly sensitive in vitro radiometric assay to analyze the distribution and regulation of brain aromatase activity. Studies using micropunch dissections revealed that the highest levels of aromatase activity are found in an interconnected group of sexually dimorphic nuclei which constitutes a neural circuit important in the control of male sexual behavior. Androgen regulated aromatase activity in many diencephalic nucleic, including the medial preoptic nucleus, but not in the medial and cortical nuclei of the amygdala. Additional genetic evidence for both androgen-dependent and -independent control of brain AA was obtained by studies of androgen-insensitive testicular-feminized rats. These observations suggest that critical differences in enzyme responsiveness are present in different brain areas. Within several nuclei, sex differences in aromatase induction correlated with differences in nuclear androgen receptor concentrations suggesting that neural responsiveness to testosterone is sexually differentiated. Estradiol and dihydrotestosterone acted synergistically to regulate aromatase activity in the preoptic area. In addition, time-course studies showed that estrogen treatment increased the duration of nuclear androgen receptor occupation in the preoptic area of male rats treated with dihydrotestosterone. These results suggest possible ways that estrogens and androgens may interact at the cellular level to regulate neural function and behavior.     

Sex-specific interactions between aggressive and sexual behavior in the rat: Effects of testosterone and progesterone

The influence of progesterone on sexual and aggressive behaviors during aggressive encounters was investigated in pairs of TP-treated male and female rats. Gonadectomized females, chronically injected with testosterone propionate (TP), showed low but consistent levels of feminine sexual behavior which alternated with aggression. Progesterone when given in addition to TP facilitated receptive and proceptive behaviors, but reduced levels of aggression. In TP-treated males, levels of aggression were the same as observed in TP-treated females. However, TP-treated males seldomly showed sexual behavior during aggressive encounters and additional treatment with progesterone did not affect their behavior. After the aggression tests, animals were tested in a social preference test in which an ovariectomized female cage mate and the opponent from the aggressive encounter served as incentives. Positive correlations between levels of aggression and social preference for an opponent were found in both sexes, although correlations only reached statistical significance when progesterone was given in addition to TP. These correlations were found in both sexes, despite the fact that group analysis revealed pronounced sex differences in social preference: males preferred to spend their time near ovariectomized female cage mates, whereas females divided their time equally among female cage mates and opponents.     

Does serotonin influence aggression? comparing regional activity before and during social interaction

Serotonin is widely believed to exert inhibitory control over aggressive behavior and intent. In addition, a number of studies of fish, reptiles, and mammals, including the lizard Anolis carolinensis, have demonstrated that serotonergic activity is stimulated by aggressive social interaction in both dominant and subordinate males. As serotonergic activity does not appear to inhibit agonistic behavior during combative social interaction, we investigated the possibility that the negative correlation between serotonergic activity and aggression exists before aggressive behavior begins. To do this, putatively dominant and more aggressive males were determined by their speed overcoming stress (latency to feeding after capture) and their celerity to court females. Serotonergic activities before aggression are differentiated by social rank in a region-specific manner. Among aggressive males baseline serotonergic activity is lower in the septum, nucleus accumbens, striatum, medial amygdala, anterior hypothalamus, raphe, and locus ceruleus but not in the hippocampus, lateral amygdala, preoptic area, substantia nigra, or ventral tegmental area. However, in regions such as the nucleus accumbens, where low serotonergic activity may help promote aggression, agonistic behavior also stimulates the greatest rise in serotonergic activity among the most aggressive males, most likely as a result of the stress associated with social interaction.     

Paternal aggression in a biparental mouse: parallels with maternal aggression

Environmental and social factors have important effects on aggressive behaviors. We examined the effect of reproductive experience on aggression in a biparental species of mouse, Peromyscus californicus. Estrogens are important in mediating aggressive behavior so we also examined estrogen receptor expression and c-fos for insights into possible mechanisms of regulation. Parental males were significantly more aggressive than virgin males, but no significant differences in estrogen receptor alpha or beta expression were detected. Patterns of c-fos following aggression tests suggested possible parallels with maternal aggression. Parental males had more c-fos positive cells in the medial amygdala, and medial preoptic area relative to virgin males. The medial preoptic area is generally considered to be relatively less important for male-male aggression in rodents, but is known to have increased activity in the context of maternal aggression. We also demonstrated through habituation-dishabituation tests that parental males show exaggerated investigation responses to chemical cues from a male intruder, suggesting that heightened sensory responses may contribute to increased parental aggression. These data suggest that, in biparental species, reproductive experience leads to the onset of paternal aggression that may be analogous to maternal aggression.